Forty years continous monitoring for bluetongue virus at an Australian site of high arbovirus activity. What have we achieved?

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1 Forty years continous monitoring for bluetongue virus at an Australian site of high arbovirus activity. What have we achieved? Lorna Melville*, Richard Weir, Neville Hunt, Steven Davis & Susan Walsh Department of Primary Industry and Fisheries, GPO Box 3000, Darwin NT 0801, Australia. * Corresponding author at: Department of Primary Industry and Fisheries, GPO Box 3000, Darwin NT 0801, Australia. Tel.: , lorna.melville@nt.gov.au. Accepted: Available on line: IV International Conference on Bluetongue and Related Orbiviruses. November 5 7, 2014 Rome, Italy Selected papers Keywords Australia, Bluetongue virus, Culicoides, Epidemiology Genetic analysis Surveillance. Summary Beatrice Hill Farm (BHF) near Darwin, Australia was identified in the early 1970 s as a site of high arbovirus activity. The first isolation of Bluetongue virus (BTV) in Australia was made on BHF in Since then, there has been continuous monitoring for BTV at BHF, the virus has been isolated on a yearly basis, with the only exception of All 10 serotypes known in Australia have been isolated at this site and an assessment of their biological behaviour made. Over the years, the methods and intensity of monitoring have been changed. In recent years molecular techniques have permitted more detailed examination of the origins of the viruses and their natural behaviour in field situations. Data collected at BHF have allowed modelling to detect likely origins of the BTVs that regularly enter Australia through wind borne infected Culicoides from South East Asia. Concurrent vector monitoring led to assess the Culicoides species more likely to be involved with transmission of these viruses. Monitoraggio del virus della Bluetongue (BTV) in un sito ad alta attività di arbovirus in Australia Parole chiave Analisi, Australia, Culicoides, Monitoraggio, Sorveglianza, Virus della Bluetongue. Riassunto Nei primi anni 70, Beatrice Hill Farm (BHF) nei pressi di Darwin, in Australia, è stata identificata come sito ad alta attività di arbovirus. Il primo isolamento del virus della Bluetongue (BTV) in Australia è stato rilevato proprio in quest area nel Da allora, la zona è stata soggetta ad un monitoraggio continuo. Il virus è stato costantemente isolato con la sola eccezione del Tutti i 10 sierotipi noti in Australia sono stati isolati in questo sito. È stato anche valutato il loro comportamento. Negli anni, sono cambiati i metodi e la frequenza del monitoraggio. Negli ultimi anni le tecniche molecolari ci hanno dato la possibilità di avere informazioni più precise sulle origini e sul comportamento del virus. I risultati ottenuti ci hanno permesso di individuare le origini dei ceppi di BTV che entrano regolarmente in Australia attraverso Culicoides infetti provenienti dal Sud-Est asiatico. Il monitoraggio di questo vettore ha permesso di determinare le specie di Culicoides più coinvolte nella trasmissione di BTV. Introduction Beatrice Hill Farm (BHF) is a Northern Territory of Australia government research farm located about 50 km South East of Darwin at S E. This area of Australia lies in the semi arid tropics and experiences hot, wet Summers and warm, dry Winters. It is within the Northern Territory of Bluetongue virus (BTV) endemic zone, which is located between approximately South. This site was identified as an area of high arbovirus activity in the early 1970 s, when sentinel herds and associated entomology studies were first established in Australia for monitoring cattle viruses. As a result of the intense entomology work at BHF, the first 263

2 Australian monitoring for Bluetongue virus Melville et al. isolation of BTV in Australia was made from a mixed pool of Culicoides collected in 1975 (St George et al. 1978). Since then, there has been continuous monitoring for BTV at this site. Monitoring cattle for BTV is supported by regular light trap collections of Culicoides. This work is largely funded by the National Arbovirus Monitoring Program (NAMP), which started in 1993 and is jointly funded by the livestock industries and state and federal governments. Materials and methods BHF sentinel herd The BHF sentinel herd consists of 24 young bovines sourced from the BTV free zone and replaced each year when there is minimal arbovirus activity. Lithium heparin and EDTA blood samples are collected each week from all animals, serum samples are collected on a monthly basis. This work is conducted under animal ethics approval number A11033 Charles Darwin University Animal Ethics Committee. Virus isolation Virus isolation is conducted in real time using 2 parallel systems. Lithium heparin blood samples are inoculated to embryonated chicken eggs (ECE) as described by Melville and colleagues (Melville et al. 2005). The ECE homogenates are passaged onto mosquito cell cultures (Aedes albopictus C6/36). The second passage uses C6/36 and Baby Hampster Kidney (BSR) (BHK21 clone) mammalian cell cultures. A third passage uses BSR cell cultures to detect any cytopathology due to virus replication. The ethylenediaminetetra acetic acid (EDTA) blood samples are centrifuged and the white cell fraction inoculated to C6/36 cells. The second and third passage uses BSR cells as described by Melville and colleagues (Melville et al. 2005). Viruses are identified by a combination of immunofluorescent antibody, real time polymerase chain reaction (qrt PCR) and virus neutralisation (VN) tests. Serology Serum samples are tested for BTV antibodies by competitive ELISA and if positive by VN tests (Melville et al. 2005). Molecular analysis With the co operation of the Australian Animal Health Laboratory, a selection of BTV isolates has been subjected to genetic analysis each year since This has identified a number of different genotypes circulating at BHF and enabled tracking of their movements (Pritchard et al. 2004). A detailed analysis of BTV 1 isolates has also been conducted to study the evolution of this virus over 30 years (Boyle et al. 2014). Entomology A light trap for insect collections is operated for 3 consecutive nights each month. Insects are collected into alcohol and the Culicoides species sorted and identified. Improvements to light trap design has seen replacement of the incandescent globe with a green LED. This in turn has required modifications to cope with the increased size of collections. Equipment was designed to enable accurate splitting of the collections (Hunt et al. 2009) and screening of the light traps was trialled to exclude large by catches. Molecular analysis of collections has also been used to identify the presence of BTV. Data management All virus isolation, serology and entomology data are entered onto a web based national database. This enables national zones to be developed for trade purposes (Cameron 2001). Dispersal modelling Atmospheric dispersal modelling was used to identify a putative source site following the introduction of new BTV serotypes in 2007 and 2008 (Eagles et al. 2014). Results Virus isolation and characterisation Eight serotypes of BTV have been isolated at BHF (serotypes 1, 3, 9, 15, 16, 20, 21, and 23) since the beginning of the program until Although the introduction of the ECE isolation system in 1986 resulted in a large increase in the number of BTVs isolated, no new serotypes were isolated for 20 years, until BTV 7 was found in 2007 and BTV 2 the following year. Table I summarises the isolation results until BTV 1 is the most commonly isolated serotype, being found in most years. The frequency of occurrence of other serotypes is highly variable. Characteristics of natural infections from have been reported in a previous study (Melville 2004). Table II shows the annual variation in infection rates and activity between 2004 and

3 Melville et al. Australian monitoring for Bluetongue virus Table I. Bluetongue virus serotypes at Beatrice Hill Farm. Serotypes , 3, 9, 15, 16, 20, 21, , , , , , 9, , , 20, , , , , , , , , 20 Table III. Bluetongue virus genotypes at Beatrice Hill Farm. Genotype Australia A 1992 Australia A/Java A 1993 Australia A 1994 Australia A/Java C 1995 Java C/Malaysia A Java C 2001 Australia A/Java C/Malaysia A Java C 2005 Java A/Java C 2006 Java C 2007 Australia A 2008 Java C/Malaysia A 2009 Malaysia A 2010 Malaysia A 2011 Australia A/ Malaysia A 2012 Malaysia A/Java C 2013 Related to Western topotype/malaysia A 2014 Australia A/Malaysia A Molecular analysis of BTV isolates from BHF has shown significant genetic variability on a yearly basis, as described in Table III. A retrospective study of the earliest isolates of BTV from BHF indicated that only 1 genotype was circulating during the period In 1992, a genotype previously found only in South East Asia was detected for the first time. Since that time, there have been Table II. Characteristics of natural Bluetongue virus infections, Detectable period of Bluetongue Sentinels viremia (weeks) serotype infected Min. Max / / / / / / / / / / / / / / / / / / / / / / / / regular incursions of previously exotic genotypes and evidence that reassortment of gene segments has occurred among different viruses circulating simultaneously. A detailed genetic analysis of BTV 1 isolates has shown 4 major periods of BTV 1 evolution. Each was marked by a period of relative stability of evolving genome segments followed by replacement and extinction by the introduction of novel genome segments (Boyle et al. 2014). Entomology Five species of Culicoides are currently recognised as proven vectors in Australia. These species are Culicoides brevitarsis, Culicoides actoni, Culicoides fulvus, Culicoides wadai, and Culicoides dumdumi. All such species have been identified at BHF. Attempts to improve surveillance in remote areas have shown that BTV can be identified in alcohol collections of Culicoides (Melville et al. 2008). Although this system is less sensitive than detection in the mammalian host, it does have application in remote areas of Northern Australia where it is impossible to maintain cattle herds. 265

4 Australian monitoring for Bluetongue virus Melville et al. Dispersal modelling For the introduction of BTV 7 in March 2007 a potential dispersal event occurred approximately 2 weeks earlier from sites across West Timor and Timor Leste, as well as 2 months prior, from the same source region. For the introduction of BTV 2 in January 2008, the modelling indicated that dispersal could have occurred 1 week earlier from a site in West Timor (Eagles et al. 2014). Discussion Over the past 40 years, the surveillance program at BHF has identified all the BTV serotypes present in Australia and documented their annual occurrence. The system is sensitive and robust for the detection of new incursions into Australia. In both 2007, when BTV 7 was first detected, and 2008 when BTV 2 was first detected, retrospective serology showed no evidence of these viruses in previous years. Similarly, serology associated with virus isolation shows that most infections are detected by weekly virus isolation. In addition to detection of new serotypes in 2007 and 2008, genetic analysis of isolates has demonstrated incursions of viruses of South East Asian origin in 1992, 1994, 1995, 2001, 2009, 2010, and These new genotypes have become established in the BHF environment for a limited number of years, before being replaced by a different genotype. Reassortant viruses have been detected on a number of occasions. A detailed analysis of BTV 1 isolates over 30 years has shown that the dominant mechanism prompting genetic diversity at BHF was the introduction of new viruses and reassortment of genome segments with existing viruses (Boyle et al. 2014). The development of qrt PCR tests for BTV group and serotypes has provided a mechanism for rapid detection of BTV in both cattle blood and insects, showing the potential to improve surveillance in Northern Australia. These techniques are being introduced as part of the routine testing of the NAMP. However, they will not replace routine virus isolation at BHF, which is designed as a surveillance system for commonly isolated arboviruses, not just BTV. Dispersal modelling has confirmed the likely origins of BTV and vector incursions into Australia. The use of an atmospheric dispersal model has confirmed the value of the BHF site as a location for intensive surveillance activities. Such modelling can contribute to the strategic use of limited surveillance resources (Eagles et al. 2014), enabling selection of the most appropriate site across the northern coastline. Acknowledgements The virus isolation, serology, and vector monitoring at BHF is funded by the NAMP. This program is managed by Animal Health Australia, with collaborative funding from the Australian livestock industries and the Australian State and Commonwealth governments. 266

5 Melville et al. Australian monitoring for Bluetongue virus References Boyle D.B., Amos Ritchie R., Broz I., Walker P.J., Melville L., Flanagan D., Davis S., Hunt N. & Weir R Evolution of bluetongue virus serotype 1 in Northern Australia over 30 years. J Virol, 88 (24), Cameron A.R Internet based management of arbovirus monitoring data. In Arbovirus research in Australia (M.D. Brown, P. Ryan & J. Aaskov, eds). Proc. 8 th Symposium, South Stradbroke Island, 3 7 July 2000, CSIRO, Brisbane, Eagles D., Melville L., Weir R., Davis S., Bellis G., Zalucki M., Walker P. & Durr P Long distance aerial dispersal modelling of Culicoides biting midges: case studies of incursions into Australia. BMC Veterinary Research, 10, Hunt N., Campbell D. & Hearnden M Handling large light trap collections. In Arbovirus research in Australia (P. Ryan, J. Aaskov & R. Russell, eds). Proc. 10 th Symposium, Coffs Harbour, September 2008, QIMR, Brisbane, Melville L.F Bluetongue surveillance methods in an endemic area: Australia. In Bluetongue, Part I (N.J. MacLachlan & J.E. Pearson, eds). Proc. Third International Symposium, Taormina, October Vet Ital, 40 (3), Melville L., Hunt N., Weir R., Davis S. & Harmsen M Results of a decade of virus monitoring of sentinel cattle in the Northern Territory ( ). In Arbovirus research in Australia (P.J. Ryan, J.G. Aaskov, T.D. St George & P.E.R. Dale, eds). Proc. 9 th Symposium, Australis Noosa Lakes Resort, August 2004, QIMR, Brisbane, Melville L., Hunt N., Bellis G. & Harmsen M Improving bluetongue virus surveillance in remote areas. In Arbovirus research in Australia (P. Ryan, J. Aaskov & R. Russell, eds). Proc. 10 th Symposium, Coffs Harbour, September 2008, QIMR, Brisbane, Pritchard L.I., Daniels P.W., Melville L.F., Kirkland P.D., Johnson S.J., Lunt R. & Eaton B.T Genetic diversity of bluetongue viruses in Australasia. In Bluetongue, Part II (N.J. MacLachlan & J.E. Pearson, eds). Proc. Third International Symposium, Taormina, October Vet Ital, 40 (3), St George T.D., Standfast H.A., Cybinkski D.H., Dyce A.L., Muller M.J., Carley J.G., Filippich C. & Frazier C.L The isolation of a bluetongue virus from Culicoides collected in the Northern Territory of Australia. Aust Vet J, 54,